CN106146667B - Exendin-4 fusion protein and preparation method and application thereof - Google Patents
Exendin-4 fusion protein and preparation method and application thereof Download PDFInfo
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- CN106146667B CN106146667B CN201510141640.9A CN201510141640A CN106146667B CN 106146667 B CN106146667 B CN 106146667B CN 201510141640 A CN201510141640 A CN 201510141640A CN 106146667 B CN106146667 B CN 106146667B
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Abstract
The invention aims to solve the technical problems of prolonging the half-life period of the Exendin-4 polypeptide and improving the stability and safety of the conventional fusion protein containing the Exendin-4 polypeptide. The scheme for solving the technical problem is to provide a novel fusion protein containing Exendin-4 polypeptide. The structure of the fusion protein is as follows: the polypeptide comprises Exendin-4, connecting peptide, Exendin-4, connecting peptide and IgG4Fc mutant from the nitrogen end in sequence. The fusion protein containing Exendin-4 provided by the invention has better stability, longer half-life and lower toxic and side effects. The fusion protein can be used for treating type I and type II diabetes and obesity, and has high clinical application value.
Description
Technical Field
The invention belongs to the field of genetic engineering medicines, and particularly relates to a long-acting Exendin-4 fusion protein, and a preparation method and application thereof.
Background
Diabetes mellitus is a chronic lifelong disease which is currently considered to be caused by heredity and acquired life habits and has become a worldwide public health problem seriously threatening human health.
The diabetes is divided into type 1 diabetes and type 2 diabetes, which are clinically marked by hyperglycemia, the blood sugar of the diabetes is increased due to the defect of insulin secretion or the defect of insulin action, the type 1 diabetes is deficient in insulin secretion caused by the immune destruction of islet β cells and depends on exogenous insulin supplementation, and the important mechanism of the onset of the type 2 diabetes is caused by insulin resistance and the relative insufficiency of insulin secretion of islet β cells, so that the cell amount of islet β is obviously reduced, and the function of residual islet β cells is obviously abnormal, therefore, the protection of β cell amount and the improvement of β cell function are particularly key for the treatment of the type 2 diabetes.
At present, oral hypoglycemic drugs are mainly used for treating type 2 diabetes, and sulfonylurea drugs such as glibenclamide, gliclazide, glipizide and the like, metformin, α -glycosidase inhibitors, insulin and the like are used for treating the type 2 diabetes.
As early as 60 s in the last century, Mclntyre and Elrick et al found that oral glucose promoted insulin secretion significantly more than intravenous injection, an additional effect known as the "incretin effect". This effect was further confirmed by studies hereafter to produce more than 50% of total insulin after meal consumption. After a normal person eats a meal, insulin secretion is promoted, which in turn causes insulin secretion to maintain normal blood glucose levels. In 1970, incretins, a hormone controlling insulin secretion, were discovered, and are intestinal-derived hormones in human bodies, and after eating, the hormones can promote insulin secretion and exert glucose-dependent glucose-lowering action. Incretins mainly include glucose-dependent insulinotropic hormone (GIP) and glucagon-like-1 (GLP-1). GIP secreted in the upper intestine and GLP-1 secreted in the lower intestine are both hormones that control insulin secretion by affecting plasma insulin levels to control postprandial blood glucose levels, the action of which is mediated by the corresponding specific receptors. GIP in the upper end of the small intestine is secreted into the blood in a large amount, which promotes insulin secretion, but GIP action is weakened after diabetes. Compared with GIP, GLP-1 secreted from the lower end of the small intestine has stronger insulin secretion promoting effect, and is characterized in that the GLP-1 is secreted immediately after eating, so that insulin secretion and biosynthesis are increased, glucagon secretion is reduced, and blood sugar is effectively reduced; can reduce gastrointestinal peristalsis, delay gastric emptying speed, increase satiety, reduce appetite, reduce food intake and reduce weight; directly reducing blood sugar; reducing triacylglycerols, free fatty acids; lowering blood pressure; protecting cardiovascular system. GLP-1 secretion is blood sugar dependent insulinotropic hormone secretion, does not increase the physiological action which does not exist in GIP such as hypoglycemia risk and the like, thereby showing the potential that GLP-1 can delay the progress of diabetes and reduce the cardiovascular complications of diabetes.
GLP-1 has β cell protecting effect, GLP-1 can act on pancreatic islet β cell, promote transcription of insulin gene, synthesis and secretion of insulin, stimulate proliferation and differentiation of pancreatic islet β cell, increase pancreatic islet β cell number, inhibit apoptosis of pancreatic islet β cell, improve insulin sensitivity, and increase glucose utilization, and GLP-1 can act on α cell, and inhibit α cell from secreting glucagon, thereby inhibiting hepatic glucose production, regulating nutrient metabolism and nutrient clearance.
GLP-1 is a member of the glucagon peptide family, expressed by the preproglucan gene, located on chromosome 17, an incretin, a peptide chain consisting of 37 amino acids GLP-1 gene is expressed in α cells of the pancreas, neuroendocrine L cells of the small intestine and hypothalamus, is synthesized mainly by L cells in the ileum, colon GLP-1 has two forms, GLP-1(7-36) and GLP-1(7-37) amide, and about 80% of the circulating activity of GLP-1 comes from GLP-1 (7-36).
However, natural GLP-1 is quickly degraded by dipeptidyl peptidase 4(DPP-4) in vivo, the DPP-4 is cut from the site between Ala8-Glu9 at the N end, and a fragment GLP-1 (9-36) generated by degradation is a GLP-1 receptor antagonist, so that the affinity of the GLP-1 and the receptor thereof is reduced to 1 percent, the half-life of the GLP-1 is only about 1-2 minutes, and the therapeutic effect can be generated only by continuous intravenous drip or continuous subcutaneous injection, which greatly limits the clinical application of the GLP-1.
According to the action targets of GLP-1 and GLP-1 receptor in treating diabetes, researchers develop three types of medicines: (1) GLP-1 analogues, which not only retain the efficacy of GLP-1, but also resist degradation; (2) a GLP-1 receptor agonist; (3) GLP-1 degrading enzyme inhibitors (DPP-4 inhibitors) can prevent GLP-1 secreted by the body from being degraded. At present, the research on the three aspects has been advanced to a certain extent.
Exendin-4 is a glucagon-like peptide 1(GLP-1) analogue, is a polypeptide hormone separated from saliva of a Greek lizard, contains polypeptide molecules with 39 amino acids, and has 53 percent homology with GLP-1 in amino acid sequence. The secondary structure of Exendin-4 is mainly divided into three parts: (1) amino acid residues at 1-8 positions of the N terminal form an irregular coil. This region is highly conserved, with only Gly2Ala with GLP-12Different, the DPP-4 can be antagonized to degrade, so the half-life is longer than that of GLP-1, (2) the middle 9-30 amino acid residues form a regular α helix, the amino acids of the side chains with opposite charges in the region are arranged alternately on the same side, the α helix is more stable than the GLP-1, (3) the C-end 31-39 amino acid residues are sequences which are not existed in the GLP-1, and the amino acid residues are folded back to α spiroScrewed up, with Trp25The subsequent research shows that Exendin-4 has the same physiological function as GLP-1 in mammals and acts on the same receptor, Exendin-4 can secrete insulin in a glucose concentration dependent mode to reduce the blood sugar level, stimulate the regeneration of pancreatic islet β cells, induce the transcription of pre-islet genes, promote the maturation and secretion of insulin, inhibit the generation of postprandial glucagon, delay gastric emptying, inhibit appetite and the like, and GLP-1 is rapidly degraded by DPP-4 no matter orally or subcutaneously injected, has a half-life of only 1-2 minutes, limits the clinical application of the GLP-1, and has a half-life of several hours, so the GLP-4 has a wide prospect in the aspect of treating diabetes.
Although Exendin-4 has the same physiological function as GLP-1 in mammals and is not easily hydrolyzed by DPP-4, so that the mammal has a longer half-life, patients still need to inject twice a day, and the compliance of the patients is poor. Therefore, the development of long-acting drugs based on Exendin-4 is a hot spot of research in the field of diabetes treatment at present.
With the rapid development of biotechnology, it has become a research hotspot in recent years to prolong the half-life of a drug in vivo and prevent the drug from being rapidly eliminated. Established techniques for increasing the half-life of drugs include chemical modification, microencapsulation, glycosylation, protease-resistant mutants, protein fusion, etc., and many long-acting protein drugs have been developed and applied in clinical therapy.
In order to prolong the half-life of the drug in vivo, the Fc fragment of immunoglobulin IgG is most widely used. IgG is the most abundant immunoglobulin in serum, and has a half-life of about 20-30 days. The reason for its good stability is mainly due to the fact that the Fc fragment of IgG binds to neonatal Fc receptors, prevents IgG from entering lysosomes and being degraded, bound in a pH-dependent manner and then recycled. Therefore, the Fc fragment of IgG is used to connect with active protein to form fusion protein, so as to raise the in vivo half-life of active protein and reach long-acting effect. However, Fc fragments of IgG1, IgG2, and IgG3 activate complement via the classical pathway, resulting in Complement Dependent Cytotoxicity (CDC); and can combine with Fc receptor on macrophage and NK cell surface to exert opsonization and Antibody Dependent Cell Cytotoxicity (ADCC). The Fc fragment-mediated CDC and ADCC activities are of no significance for the effect of extending the half-life of the drug and may lead to the generation of adverse reactions. The Fc fragment of IgG4 has the lowest complement and FcR binding activity compared to other types of IgG. However, in order to avoid the occurrence of CDC and ADCC activities, it is still necessary to mutate the Fc fragment thereof to obtain an IgG4Fc mutant. How to carry out mutation can obviously prolong the in vivo half-life period of the target protein, improve the stability of the fusion protein, and enable the obtained fusion protein to reduce CDC and ADCC activities as much as possible, and is also the key point for solving the problem.
Meanwhile, the Fc fusion protein currently on the market often adopts a target protein fused with the Fc fragment of IgG, which reduces the in vitro activity of the target protein, especially a polypeptide with a small self molecular weight.
Disclosure of Invention
The invention aims to solve the technical problems that the stability of the existing fusion protein containing Exendin-4 polypeptide is not good enough and the safety is not enough. The scheme for solving the technical problem of the invention is to provide a novel fusion protein containing Exendin-4 polypeptide, which is characterized in that the structure of the fusion protein is as follows: the Fc fragment mutant sequentially contains Exendin-4, connecting peptide, Exendin-4, connecting peptide and IgG4 from the nitrogen end. Namely, the fusion protein containing the Exendin-4 polypeptide has the structure of an Fc segment mutant of Ex-4-connecting peptide-IgG 4 from the nitrogen terminal.
Wherein, the amino acid sequence of the Fc segment mutant of the human IgG4 is shown in SEQ ID No.1, the mutation positions are Ser228Pro/Phe234Ala/Leu235Ala/△ Lys447, and the mutations can effectively reduce ADCC and CDC effects of the IgG4Fc fragment.
SEQ ID No.1:
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
Wherein, the amino acid sequence of the Exendin-4 polypeptide is shown as SEQ ID No. 2.
SEQ ID No.2:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。
The fusion protein is formed by recombining 2 Exendin-4-connecting peptides and human IgG4Fc mutant. The linker peptide between the fusion proteins needs to be of sufficient length and good flexibility to ensure that the 2 molecules linked are correctly folded and their biological activity. Typically, a flexible peptide of 5-25 amino acids is used. Linking peptides commonly used in the art may be used.
For example, the amino acid sequence of the above-mentioned linker peptide can be shown as SEQ ID No. 3.
SEQ ID No.3:GGGGSGGGGSGGGGS。
Furthermore, the amino acid sequence of the fusion protein is shown as SEQ ID No. 4.
SEQ ID No.4:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
Furthermore, the fusion protein also comprises a signal peptide at the nitrogen terminal.
Meanwhile, the invention also provides a nucleic acid for encoding the fusion protein.
Further, the nucleotide sequence of the above nucleic acid is shown as SEQ ID No.5 or a degenerate sequence thereof.
SEQ ID No.5:
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTGCGGTTATTTAT TGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCATCGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCCCATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTGC GGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCATCGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCCGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGGCAGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT。
The present invention also provides a gene vector containing the nucleotide sequence of claim 7.
The present invention also provides a host cell containing the gene vector of claim 8.
The invention also provides the application of the fusion protein, the nucleotide sequence or the gene vector in preparing a medicament for treating diabetes or obesity.
Wherein the diabetes is type 1 diabetes or type 2 diabetes.
In addition, the invention also provides a medicine for treating diabetes or obesity, which is prepared by taking the fusion protein as a main active ingredient. Namely, the fusion protein and pharmaceutically acceptable auxiliary materials are contained.
Wherein, the medicine can also comprise other medicines for treating diabetes or obesity.
Wherein the diabetes is type 1 diabetes or type 2 diabetes.
In order to better implement the present invention, the present invention also provides a method for preparing the above-described fusion protein. The method comprises the following steps: the gene coding the fusion protein is operably loaded into an expression vector, the expression vector is transferred into a host, and the fusion protein containing Exendin-4 is obtained by separating and purifying the host and/or culture supernatant after the host is cultured and proliferated.
Wherein, the expression vector in the preparation method of the fusion protein is a eukaryotic plasmid expression vector, an adenovirus vector or an adeno-associated virus vector.
Wherein, the host in the preparation method of the fusion protein is eukaryotic cell.
The separation and purification method in the preparation method of the fusion Protein comprises the steps of purifying the supernatant of a large-scale culture host by using a Protein A affinity chromatography column, and performing SP column chromatography to obtain the fusion Protein containing Exendin-4.
Obviously, the expression vector in the above method can use the commonly used eukaryotic expression vector, various host cells commonly used in genetic engineering, and the separation and purification method can also refer to the commonly used method to obtain the relatively pure fusion protein containing Exendin-4 of the present invention. The gene encoding the fusion protein can be operably loaded into an expression vector, and the expression vector can be transferred into a host, which can be referred to various gene engineering manuals and the specifications of the specifically used vector and host cell.
The fusion protein containing Exendin-4 provided by the invention is formed by fusing Exendin-4 and a specific mutant Fc fragment mutant of human immunoglobulin IgG 4. The fusion protein has better stability, longer half-life and lower toxic and side effects. The fusion protein can be used for treating type I and type II diabetes and obesity, has obvious long-acting characteristics, retains the biological activity of Exendin-4, can improve the control of blood sugar level through the secretion of insulin dependent on blood sugar, and does not cause the risk of hypoglycemia; inhibition of glucagon secretion; reducing the rate of gastric emptying; reducing body weight and increasing satiety, prolonging its half-life in serum. Meanwhile, the fusion protein has an Fc fragment mutated aiming at Exendin-4, and can reduce CDC and ADCC effects brought by natural IgG Fc to the maximum extent, thereby reducing adverse reactions of medicines, and having high clinical application value.
Drawings
FIG. 1 is a map of eukaryotic expression vector pAAV 2-neo.
FIG. 2 shows a double restriction analysis of the electrophoretogram (1% agarose electrophoresis). 1: 1Kb Ladder; 2: pAAV2-E4F4(Kpn I + Bgl II).
FIG. 3 electrophoretogram (1% agarose electrophoresis) of RT-PCR amplified IgG4Fc DNA fragment. 1: 1kb DNAsadeder maker; 2: CHO; 3: CHO-E4F 4; 4: positive control (pAAV2-E4F 4).
FIG. 4E 4F4 fusion protein 12% SDS-PAGE. 1: protein molecular weight standards; 2: E4F4 non-reduced; 3: E4F4 reduced form.
FIG. 5 in vitro activity assay results for E4F4 fusion proteins.
FIG. 6 Effect of E4F4 fusion protein on diet-induced obesity mouse body weight and food consumption. A: body weight of obese mice; b: food intake in obese mice.
Figure 7 effect of E4F4 fusion protein on diet-induced insulin tolerance and area under the curve in obese mice. A: obese mice insulin tolerance; b: area under the curve of insulin tolerance in obese mice.
Figure 8 effect of E4F4 fusion protein on diet-induced glucose tolerance and area under the curve in obese mice. A: obese mice glucose tolerance; b: area under the curve of glucose tolerance in obese mice.
FIG. 9 Effect of E4F4 fusion protein on plasma insulin levels.
FIG. 10 pharmacokinetics of E4F4 fusion protein in rat.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and examples.
The invention aims to provide a fusion protein containing Exendin-4. The fusion protein is formed by fusing Exendin-4 and an Fc fragment mutant of human immunoglobulin IgG 4. The fusion protein has better stability and longer half-life period, can obviously promote the secretion of insulin, improves the sensitivity of an organism to the insulin, and has the function of reducing weight.
Because the molecular weight of the Exendin-4 is small, the Exendin-4 can be quickly cleared by the kidney, the Exendin-4 needs to be injected twice a day to maintain the treatment effect, the Exendin-4 is limited to a certain extent clinically, and in order to better exert the summary treatment effect, the Fc fragment mutant fusion of the Exendin-4 and the human immunoglobulin IgG4 is utilized to prolong the half-life period of the Exendin-4. Different from the fusion of a target protein and IgG in the prior art, the activity of the protein is reduced, the effective dose is increased, and the risk of adverse reaction is increased. In the invention, two Exendin-4 proteins are fused with IgG4, so that the half-life of the fusion protein in vivo is prolonged, and the activity of Exendin-4 is maintained.
The invention provides a fusion protein for treating diabetes and obesity, which is formed by recombining 2 Exendin-4-connecting peptides and a human IgG4Fc mutant.
The connecting peptide is a group of flexible peptide sections without secondary structures and composed of amino acids, and the number of the amino acids is not more than 25.
The amino acid sequence of the commonly used connecting peptide is shown as SEQ ID No. 2.
The IgG4Fc mutant was prepared by mutating the complement and FcR binding sites in the Fc region by point mutation, i.e., Ser228Pro/Phe234Ala/Leu235Ala/△ Lys447. specifically, serine (Ser) at position 228 was mutated to proline (Pro), phenylalanine (Phe) at position 234 was mutated to alanine (Ala), and leucine (Leu) at position 235 was mutated to alanine (Ala), and lysine (Lys) at position 447 was deleted to ensure C-terminal uniformity.
The connecting peptide between the fusion proteins has enough length and better flexibility to ensure that the connected 2 molecules can be correctly folded and ensure the biological activity. Typically, a flexible peptide of 5-25 amino acids is used.
The fusion protein of the invention has the full length of Exendin-4-connecting peptide-IgG 4Fc mutant, and the amino acid sequence is shown as SEQ ID No. 4. The fusion protein is hereinafter referred to as rhE4F4 fusion protein.
It will be apparent to those skilled in the art, having the benefit of the present disclosure, that the present invention may be practiced using molecular biological and immunological techniques that are conventional in the art.
The following examples are provided for further details.
Example design of E4F4 fusion protein and construction of recombinant expression vector
Designing the sequence of the fusion protein, and mutating the human IgG4Fc fragment((aa.99-327) fused at C-terminal of two Exendin-4 fragments, meanwhile, in order to realize the secretory expression of the fusion protein, a mouse signal peptide sequence is added at N-terminal to obtain the final fusion protein, ssmIg-Ex-4- (G4S)3-Ex-4-(G4S)3-human mutant IgG4Fc(aa.99-327)。
The sequence of the fusion protein containing the signal peptide is as follows (SEQ ID No. 6):
MGWSCIILFLVATATGVHSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。
the corresponding coding nucleotide sequence is shown below (SEQ ID No. 7):
ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAACAGCTACAGGTGTCCACTCCCATGGTGAAGG AACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGA GGACCAAGTAGCGGGGCACCTCCGCCATCGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCCC ATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTGCGGTTATTTATTGAGTGGCT TAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCATCGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCCGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGGCAGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTTGA。
a DNA fragment (SEQ ID No.7) of E4F4 fusion protein containing a mouse signal peptide is obtained by a whole-gene synthesis method (Shanghai Yingjun company), and the synthesized fragment is directly inserted into an expression vector pAAV2-neo, and a plasmid map of the fragment is shown in figure 1. The recombinant clone is identified by Bgl II and Kpn I double enzyme digestion, and a fragment of about 1100bp is cut out, as shown in figure 2. The sequence of the DNA fragment is verified to be consistent with the designed sequence by Shanghai Yingjun company.
EXAMPLE two CHO stably expressing strains (CHO-E4F4 cells) were screened and identified
10 μ g of the pAAV2-E4F4 plasmid from example 1, prepared in large quantities, was transfected into CHO cells (available from ATCC, USA) using Lipofectamine2000 (available from Invitrogen, USA). The expression condition of the fusion protein in the culture supernatant is detected by ELISA, after four rounds of screening, E4F4 cells with high expression are preferably selected, and the cells with stable expression are named as CHO-E4F4 cells.
RNA from CHO-E4F4 cells was extracted and RT-PCR was used to identify the transcription of the insert. As shown in FIG. 3, CHO-E4F4 was a stable cell line that correctly recombinantly expressed E4F 4.
EXAMPLE preparation of the Tri rhE4F4 fusion protein
CHO-E4F4 cells were cultured in large scale, the supernatant was purified by Protein A Sepharose F.F (available from GE, USA) affinity chromatography column, and the electrophoretogram of the purified Protein is shown in FIG. 4, and the purity can reach more than 90%.
The method comprises the following specific steps:
(1) pretreatment of expression supernatant: the supernatant was slowly adjusted to pH 7.2 with 1M NaOH, centrifuged at 4000rpm for 10min at 4 ℃ and the supernatant was collected.
(2) The supernatant was subjected to Protein A Sepharose F.F. affinity chromatography, and the fusion Protein was washed off with 0.1M citric acid buffer (pH3.0), and the pH of the washed fusion Protein was adjusted to 7.2 with 1M NaOH.
(3) The resulting rhE4F4 fusion protein was dialyzed against 10mM phosphate buffer and stored at-20 ℃.
EXAMPLE four rhE4F4 fusion proteins for in vitro biological Activity assays
The in vitro biological activity of rhE4F4 fusion protein was tested using a cAMP enzyme-linked assay. The cAMP detection kit is a product of American R & D company, the operation method is carried out according to the kit specification, the cell line is rat insulinoma RIN-5F cells (purchased from American ATCC company), the subculture of the cells is carried out according to the conventional method, and the positive control is Exendin-4 (purchased from American Sigma company).
rhE4F4 fusion protein in vitro biological activity assay method as follows:
(1) inoculation of RIN/5F cells in 96-well plates 1.2X 104Cell/well, inoculating 96-well plate, standing at room temperature for 1hr, and standing at 37 deg.C and 5% C02Culturing for 72 hr.
(2) Sample dilution: diluting each sample to an initial concentration of 1 μ g/ml with a sample diluent; equal 3-fold dilutions were made from the starting concentration for 12 dilutions, 3 replicates per gradient, while setting sample dilutions and 1 × DMEMH as control wells.
(3) The supernatant from the 96-well plate was discarded, and 100. mu.l of each diluted sample was added to the 96-well plate at 37 ℃ with 5% C02Culturing for 0.5 hr.
(4) Extraction of cAMP: the cAMP detection kit of R & D lyses cells to extract cAMP, takes out a 96-well plate, discards supernatant, and washes with PBS 3 times; adding 1 Xlysate in the kit, and freeze-thawing for 3 times.
(5) Measurement of cAMP content: the lysates in the duplicate wells were mixed, and 100. mu.l of the lysate was used to detect cAMP content using a cAMP detection kit for RD.
The in vitro biological activity of the E4F4 fusion protein is determined as shown in FIG. 5, ED of Exendin-450ED of rhE4F4 fusion protein at 3.79ng/ml50It was 2.74 ng/ml. The results show that the fusion of IgG4Fc fragment did not affect the activity of Exendin-4, i.e., the ability to activate the GLP-1 receptor (GLPR).
Example preliminary pharmacodynamic study of penta rhE4F4 fusion proteins
A mouse diet induced obesity model (DIO) is established to research the treatment effect of the E4F4 fusion protein on the mouse diabetes model in vivo.
The experimental method comprises the following steps: male mice C57/B6 (purchased from beijing vingtorihua) weighing 40-45 g were selected and randomly divided into 3 groups: the normal saline group (NS group) and the E4F4 fusion protein group (E4F4 group) were fed with high-fat diet for 8 weeks to develop symptoms of obesity, insulin resistance and elevated blood sugar, and then the treatment of the mice was started. While normal mice were not fed high fat diet at the same time.
The treatment regimen was subcutaneous administration of both the normal saline group (NS group) and the E4F4 fusion protein group (E4F4 group), and 1.8mg/kg once a week for the rhE4F4 fusion protein group. The body weights and food intake of the three groups were then measured daily.
The mice were tested for insulin tolerance and glucose tolerance 14 days after treatment, and then sacrificed, orbital veins were bled, serum was isolated, and stored in a minus 80 ℃ freezer.
And (3) detecting insulin tolerance (ITT), wherein the mice are fasted for 4 hours (freely drinking water) before detection, then 0.5U/kg of insulin is injected into the abdominal cavities of the mice, tail tip blood sampling is respectively carried out at four time points of 0min, 30min, 60 min and 120min after the insulin is injected, and the blood sugar content is measured.
And (3) testing glucose tolerance (IPGTT), wherein the mice are fasted for 16 hours (freely drinking water) before testing, then 2g/kg of glucose solution is injected into the abdominal cavity of the mice, tail tip blood sampling is carried out at four time points of 0min, 30min, 60 min and 120min of glucose injection respectively, and the blood sugar content is tested.
The experimental results are as follows: the changes of the body weight and the food intake of the mice in each group are shown in figure 6, and the results show that the rhE4F4 fusion protein group, namely the E4F4 fusion protein which is administrated 1 time in a week, can obviously inhibit the increase of the body weight of the mice; and rhE4F4 fusion protein group has very significant statistical difference compared with NS group (P < 0.001); the food intake of the mice is measured every day, and compared with the NS group, the rhE4F4 fusion protein group has a certain inhibition effect on the food intake of the mice.
As shown in FIG. 7, at the end of the treatment period, the mice were fasted for 4 hours, and were injected with 0.5U/kg of insulin intraperitoneally, and blood glucose was measured at four time points of 0, 30, 60, and 120min, respectively. The experimental results show that compared with the NS group, the rhE4F4 fusion protein group can obviously improve the insulin tolerance condition, and the area under the curve (AUC) of the rhE4F4 fusion protein group and the area under the curve (AUC) of the NS group also have very significant statistical difference (P < 0.001).
As shown in fig. 7, after the treatment period of the mice is finished, the mice are fasted for 16 hours, 2g/kg of glucose is injected into the abdominal cavities of the mice, and blood sugar is detected at four time points of 0min, 30min, 60 min and 120min respectively; the experimental result shows that the insulin secreted by the rhE4F4 fusion protein group can obviously control the content of glucose and improve the glucose tolerance of a DIO mouse at 30 min; the rhE4F4 fusion protein group also had very significant statistical differences (P <0.001) compared to the NS group at three time points of 30, 60, 120 min; rhE4F4 fusion protein group also had a very significant statistical difference in area under the curve (AUC) from the NS group (P < 0.001).
The results of insulin content before and after the DIO mice are treated are shown in FIG. 9, and the results show that compared with the NS group, the insulin content is obviously reduced after the DIO mice of the rhE4F4 fusion protein group are treated, and the statistical difference is very significant (P < 0.001).
Example preliminary in vivo pharmacokinetic Studies of the hexa rhE4F4 fusion protein
SD male rats (purchased from the university of sichuan animal laboratories) at 8 weeks of age were selected and randomly divided into 3 groups: exendin-4 group, rhE4F4 fusion protein group and NS group, wherein 3 groups are injected with 100. mu.g/kg of Exendin-4, 450. mu.g/kg of rhE4F4 fusion protein and physiological saline respectively, the content change of Exendin-4 in 42 days is detected, blood is taken at different time points of 0, 1, 2, 4, 6, 14, 21, 28, 35 and 42 days, and the content of Exendin-4 in serum is detected by using an Exendin-4 detection kit (purchased from Phoenix pharmaceuticals company in America).
Exendin-4 was not detected in the NS group sera at all time points sampled; the sera from Exendin-4 group were detectable at low levels of Exendin-4 on days 1 and 2, and were not detectable at later time points. While the serum of rhE4F4 fusion protein group can be continuously detected for 35 days at the content of Exendin-4 (as shown in figure 9), the half-life t of rhE4F4 fusion protein in vivo is calculated1/2It was 6.6 days.
Claims (13)
1. The fusion protein containing the Exendin-4 polypeptide is characterized in that the structure of the fusion protein is as follows: the Fc segment mutants of Exendin-4 polypeptide, connecting peptide, Exendin-4 polypeptide, connecting peptide and IgG4 are arranged in sequence from the nitrogen end; the amino acid sequence is shown as SEQ ID No. 4.
2. The fusion protein of claim 1, wherein: also contains a signal peptide at the nitrogen terminus.
3. A nucleic acid encoding the fusion protein of any one of claims 1 or 2.
4. The nucleic acid of claim 3, wherein: the nucleotide sequence is SEQ ID number 5 or degenerate sequence thereof.
5. A gene vector comprising the nucleic acid of claim 3 or 4.
6. A host cell comprising the gene vector of claim 5.
7. Use of the fusion protein of claim 1 or 2 in the manufacture of a medicament for the treatment of type 2 diabetes or diet-induced obesity.
8. A pharmaceutical composition for treating type 2 diabetes or diet-induced obesity, characterized by: contains the fusion protein of claim 1 or 2 and pharmaceutically acceptable auxiliary materials.
9. The pharmaceutical composition of claim 8, wherein: administration is by means of aerosol, injection.
10. A method for preparing a fusion protein according to claim 1 or 2, characterized in that it comprises the following steps: the gene of the fusion protein containing the Exendin-4 polypeptide is loaded into an expression vector, the expression vector is transferred into host cells, and the fusion protein containing the Exendin-4 polypeptide is obtained by separating and purifying culture supernatant after the stable expression strain is cultured in a large scale.
11. The method for producing a fusion protein according to claim 10, wherein: the expression vector is a eukaryotic plasmid expression vector, an adenovirus vector, an adeno-associated virus vector or a lentivirus vector.
12. The method for producing a fusion protein according to claim 10, wherein: the host cell is a eukaryotic cell.
13. The method for producing a fusion protein according to claim 10, wherein: the separation and purification method is to purify the supernatant fluid of the large-scale culture host cells by a Protein A affinity chromatography column to obtain the fusion Protein containing the Exendin-4 polypeptide.
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